Process and Materials for Production of Glucosamine

Abstract

The present invention relates to a method and materials for producing glucosamine by fermentation of a genetically modified microorganism. Included in the present invention are genetically modified microorganisms useful in the present method for producing glucosamine, as well as recombinant nucleic acid molecules and the proteins produces by such recombinant nucleic acid molecules.

1 - 55 . (canceled) 
     
     
         56 . A glucosamine-6-phosphate synthase which has glucosamine-6-phosphate synthase action, said synthase being encoded by a nucleic acid sequence having a genetic modification that results in increased glucosamine-6-phosphate synthase action. 
     
     
         57 . The glucosamine-6-phosphate synthase of  claim 56 , wherein said synthase comprises at least one amino acid modification selected from the group consisting of deletion, insertion, inversion, substitution and derivatization of at least one amino acid residue. 
     
     
         58 . The glucosamine-6-phosphate synthase of  claim 56 , wherein said synthase is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:29 and SEQ ID NO:30. 
     
     
         59 . The glucosamine-6-phosphate synthase of  claim 56 , wherein said synthase comprises an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28 and SEQ ID NO:31. 
     
     
         60 . (canceled) 
     
     
         61 . The glucosamine-6-phosphate synthase of  claim 56 , wherein the genetic modification reduces glucosamine-6-phosphate product inhibition of the glucosamine-6-phosphate synthase. 
     
     
         62 . The glucosamine-6-phosphate synthase of  claim 56 , wherein the genetic modification also results in increased in vivo glucosamine-6-phosphate synthase action. 
     
     
         63 . The nucleic acid sequence of  claim 56 , wherein the nucleic acid sequence comprises a recombinant nucleic acid molecule. 
     
     
         64 . The recombinant nucleic acid molecule of  claim 63 , wherein the recombinant nucleic acid molecule is selected from the group consisting of pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149, pKLN23-151, nglmS-49 2184 , nglmS-49 1830 , nglmS-54 2184 , nglmS-54 1830 , nglmS-124 2184 , nglmS-124 1830 , nglmS-149 2184 , nglmS-149 1830 , nglmS-151 2184  and nglmS-151 1830 . 
     
     
         65 . The recombinant nucleic acid molecule of  claim 63 , wherein the recombinant nucleic acid molecule is operatively linked to a transcription control sequence. 
     
     
         66 . The glucosamine-6-phosphate synthase of  claim 56 , wherein the genetic modification increases glucosamine-6-phosphate synthase action as compared to a recombinant nucleic acid molecule encoding a naturally occurring glucosamine-6-phosphate synthase that does not have the genetic modification. 
     
     
         67 . The glucosamine-6-phosphate synthase of  claim 57 , wherein the at least one amino acid modification is at an amino acid sequence position, corresponding to amino acid sequence SEQ ID NO:16, selected from the group consisting of Ile(4), Ile(272), Ser(450), Ala(39), Arg(250), Gly(472), Leu(469), and combinations thereof. 
     
     
         68 . The glucosamine-6-phosphate synthase of  claim 57 , wherein the at least one amino acid modification is at an amino acid sequence position, corresponding to amino acid sequence SEQ ID NO:16, selected from the group consisting of:
 (a) an amino acid residue having an aliphatic hydroxyl side group for Ile(4);   (b) an amino acid residue having an aliphatic hydroxyl side group for Ile(272);   (c) an amino acid residue having an aliphatic side group for Ser(450);   (d) an amino acid residue having an aliphatic hydroxyl side group for Ala(39);   (e) an amino acid residue having a sulfur-containing side group for Arg(250);   (f) an amino acid residue having an aliphatic hydroxyl side group for Gly(472);   (g) an amino acid residue having an aliphatic side group for Leu(469);   (h) and combinations of (a)-(g).   
     
     
         69 . The glucosamine-6-phosphate synthase of  claim 57 , wherein the at least one amino acid modification is at an amino acid sequence position, corresponding to amino acid sequence SEQ ID NO:16, selected from the group consisting of: Ile(4) to Thr, Ile(272) to Thr, Ser(450) to Pro, Ala(39) to Thr, Arg(250) to Cys, Gly(472) to Ser, Leu(469) to Pro, and combinations thereof. 
     
     
         70 . The glucosamine-6-phosphate synthase of  claim 57 , wherein the at least one amino acid modification is a substitution at an amino acid sequence position, corresponding to amino acid sequence SEQ ID NO:16, of a proline residue for a leucine residue at amino acid sequence position Leu(469). 
     
     
         71 . The glucosamine-6-phosphate synthase of  claim 57 , wherein the at least one amino acid modification is at an amino acid sequence position, corresponding to amino acid sequence SEQ ID NO:16, selected from the group consisting of:
 (a) a threonine residue for an alanine residue at position Ala(39);   (b) a cysteine residue for an arginine residue at position Arg(250);   (c) a serine residue for a glycine residue at position Gly(472); and   (d) any combination of (a), (b), or (c).   
     
     
         72 . The glucosamine-6-phosphate synthase of  claim 57 , wherein the at least one amino acid modification is a substitution of an amino acid residue selected from the group consisting of:
 (a) a threonine residue for an isoleucine residue at position Ile(4);   (b) a threonine residue for an isoleucine residue at position Ile(272);   (c) a proline residue for a serine residue at position Ser(450); and   (d) any combination of (a), (b), or (c).   
     
     
         73 . The glucosamine-6-phosphate synthase of  claim 56 , wherein the increased glucosamine-6-phosphate synthase action results from the glucosamine-6-phosphate synthase having improved affinity for its substrates. 
     
     
         74 . The recombinant nucleic acid molecule of  claim 63 , wherein the recombinant nucleic acid molecule is selected from the group consisting of plasmids pKLN23-49, pKLN23-54, pKLN23-124, pKLN23-149 and pKLN23-151.